The invention refers to a new human cell line which may be used for toxicological, physiological and especially gene therapeutic examinations. Fields of application are molecular biology, medicine and pharmaceutical industry.
Gene therapy of liver diseases is an important target of molecular medicine as a multitude of genetic diseases, yet also infectious and tumor diseases, start from this organ. In the last few years, on international scale, the, first of all, expensive way of the ex vivo gene therapy was opened up, with a part of the liver being removed and the cells isolated from it being treated by the therapeutic gene in the cell culture and subsequently transplanted back into the liver. It is comparatively easy to transfer genes in cells in culture by various physical or chemical aids. Yet, the highest efficiency is reached by means of vectors coming from viruses which were modified in a purposeful way by means of gene technology. Thereby, retroviruses and adenoviruses were especially successfully used. As, on international scale, the way of ex vivo gene therapy has not proved to be sufficiently effective to allow a therapeutic effect of the gene transfer the in vivo gene transfer is the only solution. In addition to using adenoviruses which show a comparatively high affinity to liver a number of other viruses were tested as to their being suited as transfer vehicles.
Dividing differentiated hepatocytes are required for such testing. For a long time there has been tried to establish lines of differentiated hepatocytes of rodents and men.
During the last ten years there was stated that the transformation of various cell types by SV40 is a way of establishing cells which frequently results in the loss of the cell type-specific functions.
In 1986 primary hepatocytes of a rat were established as a line by means of transfection of SV40 DNA. the hepatocytes maintained their ability to produce albumin, transferrin and glucose-6-phosphatase and not to express AFP (carcino-embryonic protein). Yet, these cells showed features of transformed cells --they grew in soft agar and formed tumours in hairless mice (Woodworth et al., 1986).
In 1988 there was attempted to express the SV40 T-Ag from the embryonic liver of transgenic mice. This was an alternative method to immortalize primary hepatocytes after having been put in culture. It was assumed that the hepatocytes of transgenic mice were already immortalized in vivo. This is a possibility to avoid the loss of differentiated parameters in cultivated primary hepatocytes. The cells of the established hepatocyte lines produced albumin. They have not grown in soft agar and did not form tumours in hairless mice. This fact points to the fact that the immortalizing and transforming function of the T-Ag may be separated from each other. However, after subcloning the cells show the properties of transformed cells (Paul et al., 1988). In addition, the cells of this line coming from embryonic liver and being positive for AFP may not be regarded as differentiated hepatocytes.
In 1993 it was possible to immortalize human hepatocytes by SV40 T-Ag by means of retroviral infection. The THLE-2 and -3 lines obtained were not tumoural in hairless mice and did not express AFP. The cells of earlier passages (inoculations) were in a position to produce albumin and cytokeratin 8 (CKN8, characteristic of hepatocytes) and to metabolize a number of chemical cancerogenes. In cells of later passages the expression of cytokeratin (CKN19, characteristic of bile-duct epithelium) and a reduction of the albumin expression were observed (Pfeifer et al., 1993).
Two lines of differentiated, immortalized hepatocytes were obtained from transgenic mice who expressed the gene for the hepatocyte growth factor TGF-.alpha.. The cells of the two AML-12 and AML-14 lines were not tumoural in hairless mice and expressed mRNA for albumin, .alpha.-1 -antitrypsin (AAT) and transferrin. However, with the number of passages increasing the expression of the hepatocyte-specific genes declined (Wu et al., 1994).
To sum up there can be stated that the known liver cell lines may not be satisfactory in many respects.